Coronaviruses are a large family of viruses that typically cause mild to moderate upper respiratory tract infection. SARS-CoV-2 is a zoonotic pathogen (a virus with the ability to cross species and infect multiple hosts) and is responsible for the current COVID-19 pandemic. Many details have already been ascertained about how the virus spreads and may cause respiratory failure and sepsis in vulnerable populations. Yet, important scientific questions surrounding COVID-19 remain unanswered:
Developing COVID-19 preclinical models that closely mimic the clinical progression of the disease is vital to understand the disease and how we can treat patients effectively. The challenges in developing such models are numerous. They include infecting and replicating SARS-CoV-2 in host subjects, developing clinical characteristics of COVID-19, and ensuring that the research model is reproducible. Since in science, “all models are wrong, but some models are useful”, it is therefore essential to develop the right model to answer the right question.
Another important consideration for COVID research is to BSL3 or not to BSL3 – that is the question! Given its high virulence, an Animal Bio-Safety Level-3 (ABSL-3) facility is required to work with the SARS-CoV-2 pathogen. BSL-3 laboratories require comprehensive personal protective equipment (PPE) and strict security protocols to ensure the safety of the laboratory personnel. Working directly with dangerous pathogens like SARS-CoV-2 is necessary to understand their transmission mechanisms, their replication process, and their pathophysiological mechanisms.
Many scientists who do not have access to BSL-3 facilities are eager to join in the fight against COVID-19. While scientists who work in conventional (non BSL-3) laboratories cannot directly work with SARS-CoV-2, they can turn to different modeling techniques to replicate symptoms of the disease. Surrogate models can reproduce specific elements of COVID-19 like lung injury or pneumonia, as well as various components of the immune response that render some patients more vulnerable to COVID-19.
Researchers who have access to a BSL-3 laboratory can rely on various animal models to study COVID-19:
Mice are often a preferred mammalian biomedical research subject due to the extensive reference data found in the scientific literature, the ability to genetically manipulate their genome, their low cost, and their short lifespan. Unfortunately for COVID-19 researchers, the mouse ACE2 receptor that first binds to SARS-CoV-2 during infection is significantly different than the one found in humans. Consequently, mice do not exhibit symptoms of COVID-19 unless their immune system is compromised, or an aging model is used. Scientists, therefore, rely on various techniques to humanize the mice and make them more susceptible to the disease.
It is likely that a combination of all these disease modelling techniques will be needed to address the increasing number of questions about the virus.
Hamsters are rapidly becoming an important ally in the fight against COVID-19. Important screening endpoints in hamsters exposed to SARS-CoV-2 include infection-induced weight loss (15-20%), viral load, lung weight at necropsy, lethargy and changes in its breathing patterns. Furthermore, the diseased hamsters consistently infected co-housed naïve hamsters. All infected hamsters ultimately recovered from the disease and developed antibodies. The hamster model recapitulates many of the viral replication features and clinical symptoms sought by scientists and will likely become more prevalent in COVID-19 research laboratories.
The fight against COVID-19 will likely involve a multitude of other research models including rats, ferrets, swine and primates.
Surrogate models can help identify immune pathways, pinpoint drug targets and aid in evaluating novel therapeutic strategies. Scientists who work in conventional laboratories can also model comorbidities of COVID-19 (e.g. diabetes, obesity, ventilator-induced lung injury, bacterial infections) and develop insights that will guide clinical practice and save lives.
It is estimated that 70% of COVID-19 related deaths are attributed to respiratory failure. As such, mouse models of ARDS will certainly become more prevalent and utilized in COVID-19 research. The administration of lipopolysaccharide (LPS), whether intratracheally, systemically or via the oropharyngeal route, is one of the most common models of acute lung inflammation and ARDS in rodents. Beyond the primary LPS insult, researchers may apply additional stimuli such as prolonged or injurious mechanical ventilation to exacerbate the disease and better reproduce the human pathophysiology.
Researchers use various strains of mice for LPS modelling studies, with the C57BL/6 being the most widely published in the field. The LPS model has helped shed light on the immune pathways involved in the resolution of acute lung injuries and contributed to the development of therapeutic interventional and ventilation strategies that minimize long-term damage to the lungs. By allowing scientists to combine comprehensive hemodynamic and pulmonary mechanics parameters in different knock-out mice, the LPS model will surely help unravel some of COVID-19’s most important mysteries, even without the involvement of its main actor, SARS-CoV-2.
Coronaviruses differ considerably from influenza viruses. However, both pathogens can illicit pronounced responses in their hosts that can lead to ARDS and, on some occasions, to death. In vivo experimentation with Influenza A strain H1N1 does not require BSL-3 facilities. Influenza models of acute lung injury, coupled with a better understanding of the pathogenesis of COVID-19, may lead to novel insights about viral ARDS and how the innate and acquired immune responses and can be monitored to identify at-risk patients, and tuned to improve patient survival.
Once relevant research models are developed, the next challenge is to extract valuable biological information that can be extrapolated to humans. Measuring clinically relevant physiological outcomes such as lung function can be daunting, especially when these measurements are obtained in smaller rodents. SCIREQ offers many instruments to help non-experts acquire predictive outcomes that generate valuable information that can be disseminated to scientists and clinicians worldwide.
The flexiVent is an advanced pulmonary research platform highly adapted to assist in COVID-19 preclinical experimentation. The flexiVent measures detailed lung function outcomes in SARS-CoV-2 infected animals, LPS-induced ARDS models, genetic knock-outs and treatment groups, allowing scientists to test their hypotheses in complex organisms that closely recapitulate human biology. Software automation standardizes experimental conditions to create the highest level of reproducibility and produces comprehensive reports to easily disseminate scientific findings to the research community.
The flexiVent’s computer-programable ventilator design, scripting features and aerosol delivery capabilities are the cornerstones of numerous ARDS experiments. First, the flexiVent can synchronize the activation of an Aerogen nebulizer with the inspiratory cycle to precisely deliver aerosolized compounds such as drugs, LPS, elastase and bleomycin deep into the lungs of intubated subjects. This ventilator-assisted aerosol delivery offers a more reproducible and homogenous deposition profile than intratracheal instillation. The flexiVent can further apply a wide variety of injurious or protective ventilation modes (low or high tidal volumes, low PEEP, etc.) to reproduce clinical manifestations of ARDS and ventilator-induced lung injury in animals.
Once the appropriate model is generated, correlating COVID clinical symptoms and progression is translational measurements is key. As noted above, there are emerging reports of two phenotypes for the disease, Type L and Type H, each with specific characteristics. Type L includes Low elastance, Low lung weight, and Low gas exchange, where the lungs are behaving with nearly normal distensibility, but the patients exhibit severe hypoxemia. Type H includes High elastance, High lung weight, and High recruitment, exhibiting all the hallmarks of severe Acute Respiratory Distress Syndrome (ARDS).
The flexiVent uses the forced oscillation technique (FOT) to quantitatively measure the mechanical properties of the lungs and airways in a wide range of subjects (mice, hamsters, rats, ferrets). It offers numerous clinically relevant respiratory mechanics outcomes to phenotype COVID-19 disease models:
The combination of these outcomes provides a 360 view of the subject’s respiratory system that can help scientists develop a mechanistic understanding of the disease progression, evaluate the impact of specific genotypes and endotypes, as test novel therapeutic strategies.
The system integrates all the necessary components to simplify and automate traditionally complex inhalation studies, including aerosol generators (e.g. nebulizers, dry powder generators), atmospheric monitoring options, and in vivo/in vitro exposure sites (e.g. nose only exposure, whole-body exposure, air/liquid exposure models). The small internal volume of the system reduces ramp-up times and minimizes the amount of test article required for preclinical studies.
The compact form factor of the inExpose has enabled its use in BSL3 facilities and in standard laboratory environments. For instance, researchers in Utah relied on the inExpose nose-only towers to deliver aerosolized Rift Valley Fever Virus MP-12 (RVFV) in a BSL-3 facility. The use of the Aerogen nebulizer, coupled with automated bias flow profiles, produced consistent exposure atmospheres which resulted in uniform lung deposition and tightly-controlled mortality rates in hamsters 5-7 days post infection (p.i.). On day 2 p.i., the RVFV virus was detected in the systemic circulation of the animals, indicating that the virus was able to reach the blood-gas barrier in the distal airways. The researchers also reported that the nose-only exposure method accurately delivered viral doses and permitted better estimates of the total viral load delivered when compared to alternative approaches.
Dyspnea is a hallmark of COVID-19, especially for patients whose clinical course of the disease worsens. For these patients, shortness of breath typically sets in between the fourth and eight day of the illness and is sometimes accompanied by a loss of their sense of smell. Changes in breathing patterns have also been reported in studies involving hamsters, transgenic mice and other species infected with coronaviruses.
Breathing patterns can be quantitatively measured in preclinical subjects using instruments called plethysmographs. Plethysmographs enable the study of conscious, freely moving animals uninfluenced by anaesthetic. Plethysmographs are therefore well suited to study the respiratory drive, which depends on both the lungs and nervous system. Coupled with sophisticated breathing pattern analysis software, plethysmographs provide several respiratory outcomes such as breathing frequency, estimates of tidal volumes and other indexes that correlate with unnatural breathing (e.g. penh).
Conscious measurements are inherently more variable than invasive experiments (i.e. flexiVent) since experimental conditions cannot be standardized to the same degree. Plethysmograph-based analyses are often followed-up with more mechanistic, but invasive measurements of respiratory mechanics. Non-invasive and invasive techniques are therefore highly complimentary and can be combined to characterize the respiratory drive and mechanical properties of the lungs.
Many pharmacologists are looking towards inhaled formulations to treat COVID-19 patients with drugs, vaccines and antibodies. The respiratory route confers beneficial characteristics such as a deposition of unaltered drugs at the primary site of infection to maximize its efficiency, a reduced systemic exposure to avoid undesired side effects in other organs, and a non-invasive delivery. Unfortunately, the development of inhaled drugs is fraught with modelling challenges and high development costs which have limited a more widespread adoption of respiratory drugs.
SCIREQ and the Fraunhofer ITEM have joined forces to develop better research instruments for inhalation scientists. The ExpoCube allows researchers to study the impacts of drugs and other airborne particles on various cells (e.g. A549, Calu-3, primary human bronchial epithelial cells) and tissues (e.g. PCLS) cultured at the air-liquid interface. The innovative design of the ExpoCube permits aerosol deposition profiles that are highly efficient, reproducible and translational. Researchers can therefore explore the efficacity and toxicity of respiratory compounds and establish in vitro/in vivo correlations in a physiologically relevant model.
COVID-19 and other infectious diseases can affect multiple organ systems, often with a corresponding fever component. Digital telemetry facilitates an understanding of infectious diseases by monitoring systemic effects and responses to interventions, across small and large animal models.
In a recent interview, Brent Barre from Lovelace Biomedical Research Institute explains his current SARS-CoV-2 studies, using telemetry and plethysmography to monitor temperature fever onset and respiratory outcomes to monitor dyspnea.
These telemetry systems are appropriate for both standard laboratories and stricter BSL-3 / BSL-4 facilities, as they are often used for medical countermeasures studies, including responses to sarin exposure. Dr. Lewine’s group examined the neuroprotective effects of the NMDA receptor antagonist Ketamine following exposure. They captured EEG signals with synchronized video to quantify seizures, convulsions, and interictal spikes using rodentPACK to show the protective mechanisms of this treatment. Dr. Saxena’s group investigated the ability of serum-derived human butyrylcholinesterase, a proven effective nerve agent countermeasure, to protect minipigs against the toxic effects of high doses of sarin vapor. ecgAUTO analysis software assessed changes in ECG intervals associated with sarin-induced cardiac abnormalities.
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